- No binding
- High background signal
* Target protein not present in the cell extract, lost or distroyed during first step of preparation.
Check that the protein of interest was actually present in the cell extract/lysate. Prepare fresh extracts, do not freeze them. Use suitable protease inhibitors.
* Washes are too strong.
Decrease the amount of washing step as well as concentration of the detergent and salt.
* Large number of competing proteins are present in the sample.
Centrifugate the cell extract/lysate at 10 000 x g for 30 minutes before adding the antibody. This should help to remove unsoluble aggregates.
* Interfeering substances present in the cell extract/lysate.
Make sure that no DTT, mecraptoethanol or other reducing agents are added to your lysates. They will be the reason for antibody breakdown. Extreme pH values and increased detergent concentrations might also interfere with establishing of the antigen-antibody complex.
* Wrong beads used to isolate the antigen-antibody complex.
Check which beads Protein A and G, they will have the best binding of classes of antibodies that you are working with.
* Antibody not able to perform in immunoprecipitation or used in too low amount.
An precipitating antibody has to be changed to another one. Usually polyclonal antibodies are better in immunoprecipitation assays than monoclonal antibodies. The amount of antibody used in this assay has to be titrated. In some cases, if for instance antibodies were rised against denatured antigen, native epitopes might not be recognized.
* Is your target protein glycosylated? Glycosylation can contribute to steric hindrance of the epitope and unable the antibody to reach it, by simply masking it. You can try to denature the protein by heating up the whole extract for several minutes. Although, it will also contribute to agregate formation of other proteins present in the extract.
High background signal
* Antibody concentration used is too high.
Titrate the antibody used.
* Non-specific binding of other proteins either to the Protein A or G beads or to the antibodies.
Worth to consider in immunoprecipitation:
* SeizeTM X Immunoprecipitation Kits (Pierce)
* Dynal beads (Dynal)
Both will allow to couple the primary antibody pernamently and bypass the problem with light or heavy immunoglobulin chains which might interfere with the detection of a target protein following gel electrophoresis.